Transcript Post-translational Modification by Ubiquitin and Ubiquitin

Post-translational Modification
by UBLs
Kathleen M. Sakamoto, M.D., Ph.D.
Departments of Pediatrics, Pathology &
Laboratory Medicine,
Molecular Biology Institute, UCLA
and Division of Biology, Caltech
Ubiquitin-like modifiers
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Sumo-1*
– Discovered in 1996
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Nedd8*
– Discovered in 1997
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Apg8 and 12
– Required for autophagy
SUMO (small ubiquitin-like modifier)
Also known as Sentrin-1, PIC-1, GMP1, UBL-1, SMT3C
 Sumo-1, Sumo-2, Sumo-3
 101 amino acid protein
 Contains unique ubiquitin-homology
domains
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Homology between Ub and Sumo
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Residues 22-97 of Sumo-1 is 18% identical and 48%
homologous to human ubiquitin
Sumo-2 is a 95 aa protein 46% identical and 66%
homologous to Sumo-1
Sumo-3 is a 103 aa protein which is 97% identical to
Sumo-2 in the ubiquitin homology domain
Gly-Gly
Activation and conjugation of Sumo
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Activating enzyme complex has two subunits
– AOS1 is similar to the N-terminal half of ub E1
– UBA2 is homologous to the C-terminal half of ub E1 and
contains the cysteine necessary for linkage with Sumo
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Conjugating enzyme is UBC9
Desumoylation
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Desumoylating enzymes
– Cleave the isopeptide linkage between Sumo and
target proteins
• Ulp1
–
–
–
–
Smt3 from its conjugates
Cleaves Sumo-1 but not ubiquitin in vitro
Has both isopeptidase and C-terminal hydrolase activity
Required for G2/M transition
• Sumo C-terminal hydrolases, e.g. SENP1
Remaining questions
Function of sumoylation?
 Sumo conjugation on proteins?
 Specificity of Sumo-1, -2, and -3?
 Regulation of sumoylation?
 Other Sumo E2s?
 Sumo E3 ligases?
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Nedd8 (neural precursor cell-expressed
developmentally downregulated)
Novel mRNA enriched in fetal mouse
brain
 Developmentally downregulated
 Expression restricted to heart and
skeletal muscle
 Enriched in the nucleus
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Nedd8
Rub 1 (Yeast and plant homologues)
 Encodes a 81 amino acid polypeptide
 60% identical and 80% homologous to
ubiquitin
 Structurally more closely related to
ubiquitin

Activation of NEDD8
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Activating enzyme complex (2 subunits)
– APP-BP1 and UBA3
• The APP-BP1 N-terminal half is homologous to the Nterminal half of ubiquitin E1
• UBA3 is homologous to the C-terminal half of ubiquitin
E1 and contains the cysteine required for thiol ester
linkage with Nedd8
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Specificity is due to preferential binding of
UBA3 to NEDD8, but not ubiquitin or Sumo
Conjugation of NEDD8
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Conjugating enzyme is UBC12
– Necessary
• supported by observation in Ubc12 null yeast
mutants
– Dominant-negative form of UBC12
abolishes NEDD8 conjugation in vivo and
affects cell growth
Function of Neddylation
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Modification of E3 Cullins
– Cdc53 in yeast
– In humans, NEDD8 modifies cullin family
members
– Modifies nuclear proteins
– ?involved in centrosome cycle
Crystal structure
COP9 Signalosome (CSN)
500 kDa protein complex
 8 distinct subunits
 CSN5 or Jab1 is catalytic component of
isopeptidase
 Homology to subunits of the
proteasome (CSN5=RPN11)
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NEDD8-specific hydrolases and
isopeptidases
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Cross-reactivity with UCHs and UBPs
– UCH-L3 has NEDD8 C-terminal hydrolase
activity
– USP21 deconjugates ubiquitin and
deneddylates from NEDD8 conjugates
Remaining questions
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Is NEDD8 modification required for assembly
or function of SCF?
Role of NEDD8 conjugation of cullins on Lys
689?
How does neddylation modulate ligase
activity?
other substrates?
Role of NEDD8 or mixed ubiquitin/NEDD8
polymers?
Interferon-stimulated gene 15
Induced by IFN, lipopolysaccharides, or
upon viral infections
 E1 (ISG15 activating enzyme)
 UBP43, ISG15-specific isopeptidase
 Modified proteins: JAK-STAT proteins
 Overexpression of ISG15 leads to
neurologic disorders in mice; deletion
leads to cancer
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Autophagy
Aut7 is necessary for formation of
membrane structures in
autophagosomes
 Target is a phospholipid
 Primary amino group = e amino group of
lysine of UBL proteins
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Autophagy
Function of UBLs